CN1371282A - Drugs containing heterooligo and/or polyribonucleotides - Google Patents

Abstract

The invention relates to medicaments containing xenogenic oligo-and/or polyribonucleotides as active ingredient. The invention further relates to the use of said xenogenic oligo-and/or polyribonucleotides for the treatment of herpes virus infections and skin tumors.

Description

Drugs containing heterooligo and/or polyribonucleotides

The present invention relates to medicines containing heterogeneous oligo and/or polyribonucleotides as active ingredients, and also relates to the use of the heterogeneous oligo and/or polyribonucleotides for treating herpes virus infection and skin malignant tumors.

Background of the invention

Herpesviridae viruses are common pathogens worldwide to which most vertebrates are susceptible. The most important human herpesviruses are herpes simplex virus 1 and 2 (HSV-1, HSV-2), varicella zoster virus (VZV) and human cytomegalovirus (HCMV). HSV causes cutaneous or mucosal lesions in immunocompetitive individuals and can recur with varying frequency. The various herpes viruses are distinguished by the site of the lesion, such as cold sores or genital herpes.

Current therapeutic approaches for such viruses focus on the inhibition of viral replication, for example acyclovir is a known inhibitor of viral DNA polymerase. However, viruses can become acyclovir-resistant over time, especially for herpes simplex. In addition, although conventional drugs can provide palliation in the setting of acute lesions, they are not effective in preventing relapse.

In the late 1960s and early 1970s, it was found in transplantation studies that pretreatment of tissues with allogeneic heterologous nucleic acids or weak antigens greatly increased antibody titers in various immunoassay methods. These results were further confirmed in both in vitro and in vivo studies using a number of different antigens. However, there is no indication that nucleic acids, especially oligo- and/or polyribonucleotides of heterologous origin, are suitable for controlling viral infections.

At the same time, especially in the United States, experiments were carried out with defined synthetic poly- and oligonucleotides, especially ribonucleotides, but these experiments were not continued due to high toxicity in vivo.

It is therefore an object of the present invention to provide medicaments suitable for the treatment of herpes virus infections as well as malignant skin disorders. Furthermore, the object of the present invention is to provide a medicament for reducing the recurrence rate of skin lesions, especially those caused by viruses.

According to the invention, the object of the invention is achieved by a medicament comprising a heterooligo- and/or polyribonucleotide as active substance.

According to the invention, allogeneic means that the ribonucleotides originate from an organism different from the subject, ie the oligo- and/or polyribonucleotides do not originate from the same organism to be administered. The heterologous oligo and/or polyribonucleotides used according to the present invention are preferably from animal tissues (such as bovine tissues, fetal bovine tissues), plants and unicellular organisms, preferably from yeast cells (especially Saccharomyces cerevisiae )). Preference is given to using oligo- and/or polyribonucleotides from organisms which are evolutionarily as distant as possible from the organism being treated. Thus, in human medicine, it is preferred to use RNA from animal tissues, or particularly preferably to use RNA from plants or unicellular organisms, such as yeast.

The present invention is based on studies of herpes infection using RNA preparations. At this point it was found that the administration of isolated allogeneic RNA to skin lesions in patients with herpes simplex labialis, Herpes Simplex cruris disseminata and genital herpes simplex, in addition to the immediate effect on the lesions themselves, It also surprisingly significantly reduced relapse rates in patients with frequent relapses over many years. The RNA was then found to have similar activity against skin tumors, such as basal cell carcinoma.

The oligo- and/or polyribonucleotides used according to the invention are non-toxic and non-antigenic per se.

Formulations that effectively use total RNA and its salts and compounds are possible. Particularly preferred is tRNA. A particularly preferred way of obtaining RNA which can be used according to the invention is phenol extraction, in particular as described herein in methods I and II.

The effective amount per dose of the allo-oligo- and/or polyribonucleotide depends on various factors in each patient, such as the size and extent of the lesion site or affected area, and the type of administration. The dose range per unit dose is above 0.1 mg. The lower dose limit per unit dose is preferably at least 0.5 mg, more preferably at least 2 mg, still more preferably at least 5 mg; the upper limit is preferably 5 mg, more preferably 20 mg, still more preferably 10 mg.

The medicament of the present invention preferably contains the hetero-oligo- and/or polyribonucleotides in essentially anhydrous form, such as flakes, powders, granules, ointments and the like. However, the oligo- and/or polyribonucleotides may also be dissolved in water or another solvent.

In addition, the medicament according to the invention may contain physiologically acceptable carriers, adjuvants, diluents and/or additives and/or adjuvants.

The pharmaceutical composition containing the heterogeneous oligo and/or polyribonucleotides of the present invention can be formulated into tablets, lozenges and chewable tablets, liquid suspensions, powders, granules, emulsions, hard or soft capsules, Syrup or elixir, or extended-release dosage form or osmotic capsules for slow release.

Another pharmaceutical dosage form with particularly favorable effects is an anhydrous ointment made of PEG mixtures.

Administration is preferably by topical means, but can also be oral, parenteral, rectal or inhalational. The term parenteral here refers to subcutaneous, intravenous, intramuscular and intrasternal injection or infusion techniques.

For topical application, total RNA or tRNA powder or PEG ointment (ie anhydrous form) is preferably applied to the affected area; in the case of powder, the skin may be lightly moistened as appropriate, preferably air-dried.

A preferred embodiment of the invention is a medicament for the treatment of disorders caused by herpes viruses, which also reduces the frequency of recurrence of these disorders. The medicaments according to the invention are particularly preferably used for the treatment of lesions caused by herpes simplex virus and herpes zoster (VZV), for example lesions and relapses caused by herpes labialis (cold sores) and genital herpes simplex.

The heterogeneous oligo- and/or polyribonucleotides and drugs of the present invention are also suitable for treating malignant skin tumors, such as basal cell carcinoma.

The present invention further relates to the use of the heterogeneous oligo and/or polyribonucleotide for the preparation of medicines for treating herpes virus diseases and skin tumors.

In the case of lesion or relapse, it is preferable to treat as early as possible, and the frequency of relapse can be reduced with one dose of medication.

In addition to the treatment of humans with the allo-oligo- and/or polyribonucleotides of the invention, it is also possible to treat warm-blooded animals in this way, such as horses, cattle, sheep, etc.

The following examples and experimental results further illustrate the invention.

Example

Example 1

Preparation of oligo- and/or polyribonucleotides useful according to the invention

The relevant literature describes a large number of methods for obtaining nucleic acids, nucleotides and nucleosides, which are known to anyone having relevant experience. Two slightly modified methods are preferably used here, both of which are based on phenol treatment, method I for obtaining total RNA (Georgiev, G.P. and Mantieva, V.L. "Acta Biochemistry and Biophysics" 61, 153 (1962 )), method II is used to obtain tRNA (Bauer, S. et al. "Biotechnology and Bioengineering" 15, 1081 (1973)). Both methods are suitable for extraction of relatively large quantities.

Method I

In a Waring mixer, mix brewer's yeast (Saccharomyces cerevisiae) in buffer (A) (0.001M EDTA, 0.01M Tris-HCl buffer, pH5-6.25% sucrose, 0.5% SDS (dodecylsulfate) Sodium), a 15% suspension in 0.3% sodium deoxychlorate) was homogenized for 3 minutes at 10° C. and 3000 rpm. Mix the tissue homogenate with the same volume of solution (B) (80% recrystallized phenol in buffer (A) solution, 0.1% 8-hydroxyquinoline, 1.2% diethyl dicarbonate), and then slowly Stir for 30 minutes. All buffer solutions were prepared with deionized water previously stirred with bentonite.

The phenol-treated homogenate was then centrifuged at 10,000 g for 15 minutes at room temperature (about 20° C.). The aqueous phase was removed and the phenol and interphase discarded. The aqueous phase was mixed with the same volume of a 1:1 mixture of solution (B): chloroform/isoamyl alcohol (96:4) and extracted as described above. The aqueous phase was extracted three times with half the volume of diethyl ether in order to remove residual phenol. The solution was adjusted to contain 2% sodium acetate, and the RNA was precipitated with 2.5 volumes of absolute ethanol.

The precipitated RNA was collected by centrifugation at 0°C and 5000 rpm, and dissolved in ice-cold 0.01M Tris-HCl buffer, pH 7.0 and 0.001M MgCl ₂ . Electrophoretic pure pancreatic DNase (4 g/ml) was added to the solution, and the temperature was maintained at 22° C. for 3 hours to degrade possible DNA. Protein residues, DNase and RNase were digested with pronase (10 μg/ml) for 3 hours at 37°C. During this time, pronase is also destroyed by self-digestion. The RNA solution was extracted with solution (B) at 60°C as described above, stirred gently for 20 minutes, the phases were separated by centrifugation, the aqueous phase was removed and extracted with diethyl ether. After adding sodium acetate (final concentration 2%), the RNA was precipitated with 2.5 volumes of ethanol and centrifuged. The precipitate was dissolved in cold 2% sodium acetate, precipitated with 2.5 volumes of ethanol, and kept overnight in a -20°C alcohol mixture. The precipitate was then centrifuged, washed twice with 75% ethanol, twice with absolute ethanol, and twice with diethyl ether. After drying in the oven, dry RNA is obtained in bulk and stored in dark glass containers at room temperature.

Method II

This method is also suitable for extracting large quantities of yeast (several kilograms).

A given weight of yeast was homogenized in a quadruple volume of buffer (A) (see method I above) in a cold room. Add 40% v/v phenol solution (B) and 5% w/v ice cubes made of deionized water to the tissue homogenate and stir the mixture for 30 min. The supernatant was removed by suction filtration and then treated twice with phenol as described under Method I. The aqueous supernatant was collected in a container containing a DEAE-cellulose suspension (approximately 10% w/v, Whatman DE-22) equivalent to half the volume of the collected supernatant. Stir for 30 minutes to keep the DEAE suspension in suspension. The DEAE was then allowed to settle for one hour. Remove the supernatant by suction filtration. During this time, mix the mesophase and phenol phase with equal volumes of solution (C) (83% deionized water, 15% w/v ice cubes, 2% magnesium acetate concentrate (0.5M magnesium acetate in 0.25 mercaptoethanol)) Stir twice for 30 minutes, then separate for 70-80 minutes. The aqueous supernatant was transferred to a vessel containing DEAE, then stirred again and allowed to settle. The supernatant was taken by suction filtration, and the DEAE was washed twice with solution C as above, and then washed with solution (D) (2 volumes of magnesium acetate concentrate, 2 volumes of NaCl concentrate (3.75M NaCl aqueous solution), 0.2 volumes of Tris- HCl concentrate (2.5M Tris-HCl, pH 7.5 in water), 96 volumes of water).

The DEAE-cellulose was then applied to the column, and the bottom of the column was blocked. All the following steps were performed in a cold room at 4 °C. The column was washed with solution (D) of 12 times the content of the column at a flow rate of 1.4 l/h (only by gravity). Then use solution (E) (2 volumes of magnesium acetate concentrated solution, 0.2 volumes of Tris-HCl concentrated solution, 14 volumes of NaCl concentrated solution, 84 volumes of water, and a final NaCl concentration of 0.525M to elute tRNA at a flow rate of 3 l/h. 35A _260nm unit/ml fraction was precipitated with 1.5 volumes of ethanol.The further operation was the same as method I.

Alternatively, the final precipitate is dissolved in water and lyophilized.

A variant of this method is common phenol treatment of the starting material: crude tRNA is precipitated from the upper phase with isopropanol. After centrifugation, the precipitate was extracted with sodium acetate buffer and subjected to DEAE-cellulose chromatography. Gradient elution with sodium acetate/sodium chloride is known to those skilled in the art. See above, determination of suitable fractions by means of measurements of quotients, combined. The tRNA is precipitated with ethanol, the pellet is dissolved as above, and preferably lyophilized.

Total RNA and tRNA were analyzed for purity and identified using the following assays.

Protein according to Lowry, OH et al. (Journal of Biochemistry 193, 265 (1951)) and via A ₂₆₀ /A ₂₈₀ ≌2, DNA according to Dische ("Microchemistry" 8, 4 (1930)), total RNA according to Mejbaum ( Physiology and Chemistry 258, 117 (1939)), quantitative determination of tRNA and amino acid building blocks according to Sprinzl and Sternbach (Methods in Enzymology 59, 182 (1979)), toxicity according to M. Noeldner (personal communication), in vitro Pyrogen deficiency was determined according to DAB 1997 (LAL assay), pyrogen deficiency in vivo was determined according to Ph.Eur./DAB 1997.

Analysis results:

(Properties of total RNA and tRNA, mean of ten experiments)

Absorbance

A ₂₆₀ /A ₂₈₀ ≌1.94-2.0

C, H, N Analysis C 32.67 32.42H 5.22 5.20N 2.29 2.00

Corresponding values for various total RNA and tRNA.

UV and IR Spectroscopy

UV vs. IR spectrum varies from biological substance, they are almost the same but not identical.

molecular weight

Total RNA and tRNA from yeast ≌average 22000-27000 Daltons, vary with different preparations; protein DNA (total content) 2.3% neg. total of Saccharomyces cerevisiae

RNA 1.9% neg. Saccharomyces cerevisiae

                                                                                                                                                          

Average for average average quality. Increased purity did not result in a significant increase in therapeutic effect, not justified by the higher cost.

Regarding the amino acid structural unit of tRNA, the average of 10 analyzes Lysine 69-85 pMol/A ₂₆₀ units Phe 41-55Ser 39-50Val 77-90

These averages vary from batch to batch of yeast, varying within the stated range.

toxicity

Acute toxicity test in mice: Animals: NMRI mice, male, Janvier, France Administration: Tail vein observation period: 24 hours Random sample number: n=10 at the highest concentration Determining substances: bovine total RNA

            

tRNA solvent for cerevisiae)): 0.9% NaCl water p.i. solution Results:

Up to the maximum dose of 1 g/kg/10 ml i.v., the animals used in the test showed no prominent features during the 24 hour observation period.

lack of pyrogens

A. The pyrogenic content of total RNA and tRNA was determined by in vitro assay of endotoxin according to DAB 1997 (LAL test) and also by rabbit according to Ph.Eur./DAB 1997, as described previously.

1. Total RNA endotoxin standard EC5 amebocyte lysate-nominal sensitivity: 0.06EU/ml-measured sensitivity: 0.06EU/ml test solution: 100mg RNA in 20ml water-LAL solution (0.5%) result: water-LAL Endotoxin content of 0.5% test solution diluted 1:5: <0.03EU/ml

2. tRNA endotoxin standard EC5 amebocyte lysate-nominal sensitivity: 0.06EU/ml-measured sensitivity: 0.06EU/ml test solution: 20ml water-LAL solution (0.5%) of 100mg RNA result: water-LAL press Endotoxin content of 0.5% test solution diluted 1:10: <0.03EU/ml

B. In vivo pyrogen deficiency test according to Ph.Eur./DAB 1997

1. Total RNA test solution: 1% pyrogen-free water p.i. solution of the measured substance Dose: 1.0ml/animal Animal: 3 rabbits, in line with DAB 1997 Results: The sum of the temperature differences of the 3 rabbits is 1.05°C, so the pyrogen is not detectable.

2. tRNA test solution: 1% pyrogen-free water p.i. solution of the measured substance Dose: 1.0ml/animal Animal: 2 times 6 rabbits, in line with DAB 1997 Results: a. The sum of the temperature differences of 6 rabbits: 5.40℃

b. The sum of the temperature differences of 6 rabbits: 4.10°C, the pyrogen can be detected

Example 2

Detection of substance efficacy of the present invention

Of the 70 patients, 40 with herpes simplex type I (cold sores) and 30 with herpes simplex type II (genital herpes), both with frequent relapses, were treated with total RNA. RNA was obtained from extracts of bovine fetal tissues, except liver. Apply powdered RNA to the slightly moist lesion at a dose of 5 to 10 mg, depending on the size of the lesion, and allow to dry naturally. All patients were observed for 1 year.

Five patients did not relapse and seven patients could not be analyzed because of insufficient compliance. All other patients who had always had several relapses per year showed a significant reduction in relapses. Evaluation was performed by means of the nonparametric Mann-Whitney U test. Results were significant at p<0.001 (SPSS, Npar, Mann-Whitney U test).

In a double-blind study with an observation period of 1 year, two groups of 100 patients were tested as above with bovine total RNA or with tRNA from Saccharomyces cerevisiae, suffering from herpes labial and genital herpes simplex with 4 relapses per year above. Evaluation was performed after one year with the program SPSS, Npar test: Mann-Whitney and ^χ2 test.

The reduction in relapse compared to placebo-dosed patients was highly significant: p<0.001 in both cases. There was little difference between the two RNAs.

These results justify the use of the RNA in patients, especially without any side effects or symptoms of toxicity observed over several years.

When the substance was administered to herpes simplex patients who also had basal cell carcinoma of the face, it was found that the basal cell carcinoma was attenuated. Therefore, the indications of the medicament of the present invention also include malignant tumors.

Claims (14)

1, medicine especially for treatment herpesvirus infection and/or cutaneous tumor, is characterized in that it comprises allosome widow and/or polyribonucleotide as active substance.

2,, it is characterized in that it comprises the physiology in addition and goes up acceptable carrier, auxiliary agent, diluent and/or additive as claim 1 medicine required for protection.

3,, it is characterized in that this active substance comprises from animal tissue, plant and/or monadic widow and/or polyribonucleotide as claim 1 or 2 medicines required for protection.

4,, it is characterized in that this active substance comprises widow and/or the polyribonucleotide from yeast cells as claim 3 medicine required for protection.

5,, it is characterized in that this active substance comprises allosome tRNA as any aforementioned claim medicine required for protection.

6,, it is characterized in that this active substance comprises by resulting allosome widow of phenol extraction and/or polyribonucleotide as any aforementioned claim medicine required for protection.

7,, it is characterized in that this allosome widow and/or polyribonucleotide derive from evolution and go up away from being treated biological biology as any aforementioned claim medicine required for protection.

8,, it is characterized in that this widow and/or polyribonucleotide exist with anhydrous form as any aforementioned claim medicine required for protection.

9,, it is characterized in that it is to exist with the form that is suitable for topical as any aforementioned claim medicine required for protection.

10, the purposes of allosome widow and/or polyribonucleotide is used for the treatment of herpesvirus infection and/or cutaneous tumor.

11,, be used for the treatment of the skin and/or the mucosa pathological changes that cause by herpes simplex virus and/or varicella zoster virus as claim 10 purposes required for protection.

12, as claim 10 purposes required for protection, be used for the treatment of basal cell carcinoma.

13, the purposes of allosome widow and/or polyribonucleotide is used to prepare the medicine for the treatment of herpesvirus infection and/or cutaneous tumor.

14, the Therapeutic Method of herpesvirus infection and/or cutaneous tumor is characterized in that effective dose with per unit dosage is 0.1mg and above allosome widow and/or polyribonucleotide patient or the animals administer to this treatment of needs.